Restricted feeding is well known to affect expression profiles of both clock and metabolic genes. However, it is unknown whether these changes in metabolic gene expression result from changes in the molecular clock or in feeding behavior. Here we eliminated the daily rhythm in feeding behavior by providing 6 meals evenly distributed over the light/dark-cycle. Animals on this 6-meals-a-day feeding schedule retained the normal day/night difference in physiological parameters including body temperature and locomotor activity. The daily rhythm in respiratory exchange ratio (RER), however, was significantly phase-shifted through increased utilization of carbohydrates during the light phase and increased lipid oxidation during the dark phase. This 6-meals-a-day feeding schedule did not have a major impact on the clock gene expression rhythms in the master clock, but did have mild effects on peripheral clocks. In contrast, genes involved in glucose and lipid metabolism showed differential expression. In conclusion, eliminating the daily rhythm in feeding behavior in rats does not affect the master clock and only mildly affects peripheral clocks, but disturbs metabolic rhythms in liver, skeletal muscle and brown adipose tissue in a tissue-dependent manner. Thereby, a clear daily rhythm in feeding behavior strongly regulates timing of peripheral metabolism, separately from circadian clocks. Daily recurring biological events in the mammalian behavioral system such as the sleep-wake, feeding-fasting and rest-activity cycles are under the control of the master or central clock, located in the suprachiasmatic nucleus (SCN) in the anterior hypothalamus of the brain. The SCN clock output controls and coordinates other secondary clocks in the brain and peripheral clocks present in virtually every other tissue in the body through neural, humoral and behavioral rhythms, and consequently also regulates bodily rhythms in metabolism ,2,3,4. TCircadian disruption, for example, by performing shift work, can thus be expected to have adverse effects on energy homeostasis. Indeed, shift work in humans, as well as in animal models of shift work, shows disturbances in energy metabolism and an increased risk for metabolic disorders such as obesity and type 2 diabetes (T2DM) , respectively; independent t-test p < 0.0001, ad libitum fed animals  was abolished in the 6-meal group . Body weight gain after 6 weeks was lower for the 6-meal group as compared to the ad libitum fed group , but not in the 6-meal animals    .The daily pattern of RER was altered in the 6-meal group, not showing a clear cosinor-like rhythm alike the  the day a. These = 0.354) b. This d< 0.001) . Moreove 0.0581) c. Two-waad libitum group, showing a clear L/D difference, although the 6-meal group also showed 6 peaks in activity over 24 h . A paired t-test confirmed the significant effects of L/D in both the ad libitum and 6-meal group (p < 0.0001 for both groups), with higher locomotor activity during the dark period in both groups   .The daily pattern of locomotor activity for the 6-meal group was very similar to that of the ver 24 h a. These h groups b. Cosinoctively) . Total l= 0.538) c. Two-waad libitum group, showing a large L/D difference, although the 6-meals-a-day group had 6 clear peaks in energy expenditure throughout the day , although again with a somewhat smaller amplitude in the 6-meals-a-day animals   (p < 0.001) and Feeding × L/D (p < 0.001), but no effect of Feeding (p = 0.165).The daily pattern of energy expenditure for the 6-meals-a-day group was similar to the  the day a. These  animals b. Additictively) . Energy = 0.136) c. Two-waad libitum group, showing a clear L/D difference . Cosinor analysis further revealed that there were no significant differences in amplitude or acrophase between the two groups   c. ad libitum and 6-meals-a-day feeding conditions. Two-way ANOVA analysis showed a significant effect of Time for both Per1 and Per2 (p < 0.001). Cosinor analysis confirmed the rhythmicity of both clock genes , but not in the ad libitum (p = 0.313) group  group C.ad libitum and 6-meals-a-day feeding conditions. Two-way ANOVA showed a significant effect of Time (p < 0.001). Cosinor analysis detected no significant rhythmicity in the 6-meals-a-day groups (p = 0.139) and a marginally significant rhythm in the ad libitum group (p = 0.054)  D.p < 0.001) for all clock genes except Cry2. A significant interaction between Time and Feeding was found for Per2, Rev-erbα and Dbp . Cosinor analysis revealed no shift in expression for any of the clock genes when comparing ad libitum with 6-meals-a-day feeding except for Bmal1 . Mean expression levels were affected for Bmal1 and Cry1 and the amplitude was dampened for Bmal1, Cry1, Per2 and Rev-erbα . Additionally, a significant effect of Time was found for all the studied clock genes as well as an Interaction for Cry1, Per2, and Rev-erbα . Pdk4 and Fgf21 lost rhythmicity, but Lpl and Srebp1c gained rhythmicity. The other metabolic genes tested  were not rhythmic in either condition  on energy metabolism by focusing on (clock) gene expression in liver, skeletal muscle, BAT and WAT ,32,35. SAbolishing the daily feeding-fasting cycle with an ultradian 6-meals-a-day feeding schedule  without caloric restriction did not affect the SCN clock, and only slightly affected peripheral clock gene expression rhythms in liver, BAT and SM with small changes in acrophase, mesor and amplitude levels for some clock genes in a tissue- and gene-dependent manner. On a whole-body level, minor changes in the daily rhythms in locomotor activity and energy expenditure were found, but the daily rhythm in RER was severely disturbed in the animals on the 6-meals-a-day feeding schedule. In agreement with the disturbed daily RER pattern, eating according to the 6-meals-a-day schedule especially affected expression profiles of genes involved in lipid metabolism in liver, BAT and SM. Hence, in rats, 6-meals-a-day feeding without caloric restriction does not severely impact the molecular clock, but does induce phase changes in the daily rhythmicity of the respiratory quotient, i.e., affect metabolic flexibility.ad libitum condition, they were not under hypocaloric conditions, as they continuously gained weight throughout the experiment. Nevertheless, at the end of the experiment, body weight was slightly lower when compared to ad libitum conditions. This is similar to what was observed in previous studies using the 6-meals-a-day feeding schedule in rats [To successfully eliminate the daily rhythm in feeding behavior we reduced the time of food access, rats could eat ~1.3 h each day evenly spread across the 24-h period. An important issue that arises with this experimental design, especially when investigating metabolism, is that the experimental group eats less and potentially becomes hypocaloric . Importantly, although in our experiments the animals on the 6-meal regimen ate less compared to the  in rats ,34,39,40 in rats . Additioad libitum conditions. Even more surprising, maybe, its acrophase showed a 10-h phase advance, with highest RER values found when they are low in ad libitum conditions and lowest values when they are highest during ad libitum conditions. These data show that metabolic rhythms and metabolic flexibility may be severely disturbed, even though the circadian system itself is hardly affected.During the 6-meals-a-day feeding regimen, the animals largely maintained the day-night rhythm in locomotor activity that is often lost or even inverted to the inactive phase during light phase TRF ,28. DespEnergy expenditure in 6-meals-a-day feeding rats showed a sudden rise during each meal access, especially during the light period. This rise in energy expenditure during each meal may be due to meal induced thermogenesis, but it is also strongly correlated with the rise in locomotor activity during each meal access. These rises in activity were most obvious during the light period, i.e., the normal sleep period. Inherently, this paradigm could also potentially alter the sleep/wake cycles of the animals, but this remains to be tested. The SCN clock synchronizes the other brain and peripheral clocks. Time-restricted feeding entrains the peripheral clocks, but not the SCN clock. Intriguingly, restricted feeding coupled to caloric restriction does shift main circadian clock output measures  in rats and shifts the SCN clock in mice ,41,42, aPeripheral clocks were affected by the 6-meals-a-day feeding paradigm in a tissue-dependent as well as a clock gene dependent manner. For example, rhythmic expression of Per1 in SM and Cry2 in liver was lost under the 6-meals-a-day regimen, while Dbp in BAT  and Dbp Under the 6-meals-a-day feeding regimen Srebp1c lost its rhythmic expression, while Pgc1α gained rhythmicity upon 6-meals-a-day feeding. Several metabolic genes also showed a trend for changes on average expression levels as a result of the 6-meal feeding regimen  or showed a significant phase shift of their acrophase (Pdk4 and Ucp3). This effect of 6-meals-a-day feeding on genes involved in lipid metabolism is in line with our previous observation of increased plasma leptin levels . Finallyad libitum fed group. As expected, a peak in RER occurred almost immediately after each feeding session in the 6-meals-a-day experiment, regardless of the time of day  that have been found to be activated by prolonged fasting ,51. Intead libitum fed animals during the daytime might disturb the rhythmic expression of these metabolic genes. Elimination of this fasting period through the 6-meal paradigm potentially unmasks this disturbance. In line with this, eliminating the rhythm in feeding behavior only marginally affected the peripheral clocks but did lead to altered expression of metabolic genes. It thus might be possible that the feeding behavior  itself suppressed the rhythmic control of the peripheral clock on these peripheral metabolic genes. This would thus imply that some metabolic genes are mainly regulated by the feeding behavior while other metabolic genes are controlled by the combination of the peripheral clock and feeding/fasting behavior. In line with this is a study with clockless (Cry1/2 KO) mice, showing that for hundreds of metabolic genes in the liver, rhythmic expression could be restored by enforcing TRF [ad libitum group while for the 6-meal group this rhythm is found, for example, through small shifts in the expression profile or a slightly altered amplitude a gene could be detected to be rhythmically expressed in the 6M group, but not in the ad libitum group.In all three tissue types investigated here, an apparent gain of rhythmicity was found for one or more metabolic genes in the 6-meal group. Although this might seem counter-intuitive, as the rhythm in feeding behavior was abolished, there are several possible explanations for these findings. The natural fasting period of cing TRF . HoweverLastly, we do not believe that the duration of our intervention is too short to notice more substantial changes in gene expression profiles and metabolic phenotype. Our study protocol lasted for 6 weeks, and therefore exceeds the duration of most TRF protocols, which generally last between 1 and 4 weeks.An increasing amount of evidence suggests that not only diet composition, but also the timing of food intake can both improve or deteriorate energy homeostasis. Recently, several studies in humans have tried to identify potential mechanisms that can explain these links between rhythmic aspects of (feeding) behavior and metabolic diseases, such as obesity and T2DM. One such study found a positive correlation between the fragmentation of daytime activity rhythms and occurrence of obesity and central adiposity in European adolescents . Similarad libitum control group. Mice that consumed isocaloric meals twice a day had lower lipid levels, suppressed gluconeogenesis, increased leptin sensitivity, increased muscle mass and decreased adiposity, all suggesting that consuming multiple distinct meals with an equal interval of intermeal fasting, without caloric restriction, can prevent metabolic disorders such as T2DM and obesity. Another possible explanation for these findings is the clear fasting period in the experimental group. Several studies show beneficial effects of (prolonged) fasting on metabolism and health in both humans and rodents . One suiewed in . After tfat diet . Contrasfat diet ,59. Howerk phase . AdditioF itself . RecentlF itself ,63,64,65F itself . Even thF itself ,68. Thusad libitum access to tap water. Animals only received chow food . Animals were habituated to the 6-meals-a-day feeding regimen by having access to their food bin 6 times a day, temporally spaced equally over the 24 h light/dark (L/D)-cycle once every 4 h. In this way there were three feeding opportunities  during the light period at ZT2, ZT6 and ZT10, each meal consisting of 12 min of food access and three feeding opportunities during the dark period at ZT14, ZT18, and ZT22 each consisting of 11 min of food access, as the animals eat at a slightly higher pace during the dark period. All rats learned within two weeks to eat equal amounts of food in the light and dark period under this feeding regimen. Animals in the control group under ad libitum conditions had free access to food 24 h/day. After 4 weeks, a subset of animals from both the 6-meal schedule (n = 15) and the ad libitum group (n = 16) were placed in metabolic cages for 4 days in order to measure physiological and metabolic parameters, while remaining on their assigned feeding conditions. After 6 weeks, the rats were sacrificed at ZT2, ZT8, ZT14 or ZT20 and soleus muscle, liver and BAT tissues were carefully collected, snap frozen in liquid nitrogen and stored at −80 °C until RNA isolation was performed. Brain tissue was collected from two independent experiments with identical experimental procedures. In this way, we could store brain tissue both freshly frozen for use with in situ hybridization as well as store brain tissue post-fixed with 4% paraformaldehyde for immunohistochemistry (IHC). Liver tissue and frozen brain tissue came from one set of animals (n = 33), soleus muscle, BAT, PFA fixed brain tissue and metabolic measurements came from the other set of animals (n = 31). All experimental procedures were approved by the Animal Ethics Committee of the Royal Dutch Academy of Arts and Sciences  on 23 September 2010 (Ethic Code: NIN2010.44) and in accordance with the guidelines on animal experimentation of the Netherlands Institute for Neuroscience. Sixty-four male Wistar WU rats, 7 weeks of age upon arrival at the institute , were used for the experiments. During the experiment, all animals were housed individually in a controlled climate environment of 21 °C and under 12:12 light:dark conditions for the entire experiment, with Zeitgeber time (ZT) 0 being the time of lights on and ZT12 being the time of lights off. All animals had ad libitum or 6-meals-a-day feeding schedule conditions. After a day of acclimatization to this new environment, the parameters for food intake, locomotor activity, respiratory exchange ratio (RER) and energy expenditure were measured for three consecutive days (72 h). Metabolic PhenoCages (TSE systems) were used to measure several metabolic parameters, while animals remained on their In a subset of animals from both groups, a temperature logger  was placed subcutaneously during a short isoflurane anesthesia  at the lumbar back region in order to prevent interference of the signal with BAT activity. After a recovery period of 9 days body temperature was measured every 15 min for three consecutive days.Soleus muscle tissue was mechanically homogenized while kept on dry ice. The BAT tissue and liver were crushed in Trizol by using a homogenizer machine. For all the tissues, RNA isolation was done using the NucleoSpin RNA isolation kit . For muscle RNA isolation, three additional washing steps with 75% ethanol were performed. RNA was eluted from the spin column using 40 μL of H2O and RNA concentration and quality of the RNA were determined using a DS-11  spectrophotometer and a nanochip using Agilent 2100 Bioanalyzer , respectively. Although RNA integrity number (RIN) values above 5 were considered acceptable, all samples had a RIN above 8.Two hundred ng from both muscle and liver, 350 ng from BAT of isolated RNA were used as input template for cDNA synthesis. The Transcriptor First Strand cDNA synthesis kit  was used with oligo-dT primers  was used as negative control to check for genomic DNA contamination during the RT-qPCRs. RT-PCRs were run using an UNO-Thermoblock .One to nineteen (1:19) diluted cDNA was used for all qPCRs to detect muscle, BAT and liver gene expression profiles. Expression levels of all genes were standardized by dividing over the geometric mean of two or three housekeeping genes: TBP, GAPDH and Cyclophilin for muscle; TBP, HPRT1 and GAPDH for BAT and HPRT and GAPDH for liver. RT-qPCR was performed using a LightCycler 480 (Roche). Different housekeeping genes were used for the different tissues as feeding rhythms are known to affect commonly used housekeeping genes in a tissue-dependent manner and thus need to be tested for each different tissue to determine their suitability . ExpressAfter decapitation, fresh brains were stored in −80 °C before cryo-sectioning was performed. The transverse sections (20 μm) covering the rostro-caudal axis to the SCN were cut and collected on frosted glass slides in series of 6 sections. Slides were stored at −20 °C until experiments were performed. Antisense and sense RNA probes were generated with an in vitro transcription kit . Here we used riboprobes of rPer1, rPer2  and rAvp . Hybridi2O2 in TBS (30 min) to suppress endogenous peroxidase activity, thereby reducing background staining. Again, brain sections were rinsed in TBS. Brain sections were then transferred in a solution containing 10% normal serum goat according to the host species of the primary antibody) and Triton X-100 (0.1%) in TBS for 2 h, followed by incubation in the primary antibody (24 h at 4 °C). We used rabbit polyclonal anti arginine-vasopressin (AVP) . The sections were washed in TBS, then incubated (2 h at 4 °C) with biotinylated goat anti-rabbit IgG  for AVP immunostaining. After this, sections were rinsed in TBS and incubated (2 h) in a solution containing avidin–biotin peroxidase complex . Following incubation with ABC reagents, sections were rinsed 4 times in PBS, and incubated with H2O2  and 3,3′-diaminobenzidine tetrahydrochloride  diluted in water. Thereafter, sections were rinsed with TBS, wet mounted on slides coated with gelatin, dehydrated through a series of alcohols, soaked in xylene, and cover slipped. Photomicrographs were taken on a Leica DMRB microscope  with an Olympus DP50 digital camera . The number of immune-positive cells was counted on one medial section in both SCN’s and averaged.After decapitation, brains were dissected and post fixed in 4% paraformaldehyde for 24 h in a 4 °C cold room. Following the 24 h post fixation the brain tissue was transferred again to fresh 4% paraformaldehyde for the next 48 h followed by cryoprotection in 30% sucrose solution in a 4 °C cold room until sectioning was performed. Five series of 30 μm coronal SCN sections were prepared on a cryostat and collected in the cryoprotectant and washed with 1× Tris Buffer Saline pH 7.6 (0.1 M TBS). Subsequently, sections were incubated in 3% Ht-tests and two-way analyses of variance (ANOVA) were performed on parameters obtained from cosinor analysis , and gene and protein expression profiles. Two-way ANOVAs (repeated measures) were performed to assess the effects on the physiological measures  using GraphPad Prism 7. Also (paired) t-tests to determine differences between the light and dark phase, as well as differences in 24 h physiological measures between the ad libitum and 6M group were performed using GraphPad Prism 7. Finally, Pearson correlations coefficients between the different clock and metabolic genes of the peripheral tissues were also calculated using GraphPad Prism 7.All data are presented as means ± SEM. Daily rhythmicity of gene and protein expression profiles were assessed using cosinor analysis determining mean, amplitude and acrophase of the considered measures with SigmaPlot 13 software . Data were fitted to the following regression: y = A + B·cos(2π(x − C)/24), where A is the mean level, B the amplitude and C the acrophase of the rhythm, using SigmaPlot 13 software. Statistical analysis using Using our 6-meals-a-day feeding regimen to eliminate the daily rhythm in feeding behavior, we observed that this ultradian feeding behavior has no effect on the SCN clock, and has limited effects on peripheral clock gene rhythms in liver, BAT and SM, as long as the animals do not become hypocaloric. These results indicate that other, environmental or endogenous cues clearly also are involved in the control of these peripheral rhythms. The lack of effect of the 6-meals-a-day feeding paradigm on the central pacemaker in the SCN confirms its rigidity to the synchronizing effects of light. However, this does not necessarily prevent misalignment between peripheral clocks and the central clock or between peripheral clocks themselves, because the changes in gene expression were often regulated in a tissue-dependent manner. Finally, the observed changes concerned mainly genes involved in lipid metabolism in either liver or eWAT (,41, pres


Edwardsiella ictaluri is a Gram-negative intracellular pathogen that causes enteric septicemia of catfish (ESC). Successful vaccination against intracellular pathogens requires T cell priming by antigen presenting cells (APCs) that bridge innate and adaptive immunity. However, the evidence on immunological mechanisms that underscore E. ictaluri pathogenesis and the protective role of live attenuated vaccines (LAVs) is scarce. We assessed the expression of immune genes related to antigen presentation by real-time PCR and the distribution patterns of Langerhans-like (L/CD207+) cells by immunohistochemistry in the immune-related tissues of channel catfish challenged with two novel E. ictaluri LAVs, EiΔevpB, and ESC-NDKL1 and wild type (WT) strain. Our results indicated significantly elevated expression of IFN-γ gene in the anterior kidney (AK) and spleen of vaccinated catfish at the early stages of exposure, which correlated with increased numbers of L/CD207+ cells. In general, the ESC-NDKL1-induced IFN-γ gene expression patterns in the AK resembled that of the patterns induced by EiΔevpB. However the MHCII gene expression patterns differed between the strains with significant increases at 6 h post-challenge (pc) with the EiΔevpB and at 7 d pc with the ESC-NDKL1 strains, respectively. Significant increases in activity of T helper type polarization genes such as IFN-γ and T cell co-receptors after exposure to ESC-NDKL1, in combination with elevated numbers of L/CD207+ cells at 7 d pc with both LAVs compared to uninfected and the WT-exposed counterparts, were documented in the spleen. The dominant pro-inflammatory environment with dramatically overexpressed inflammatory genes in the AK and 7 d pc in the spleen in response to E. ictaluri was found in exposed catfish. In general, the pro-inflammatory gene expression profiles in the ESC-NDKL1 pc showed more similarities to the WT strain-induced gene profiles compared to the EiΔevpB counterpart. In addition, E. ictaluri WT significantly decreased the numbers of Langerhans-like L/CD207+ cells in the AK and spleen at 3 and 7 days pc. In conclusion, we report the differential framework of initiation of innate and adaptive immune responses between E. ictaluri strains with both LAVs having a potential of satisfying the stringent requirements for successful vaccines. Edwardsiella ictaluri  is a Gram-negative facultative intracellular pathogen that causes enteric septicemia of channel catfish (ESC), one of the most devastating diseases in the US catfish industry  against ESC was developed by Klesius and Shoemaker, and this vaccine can provide efficient protection to juvenile catfish  was developed by Wise et al. and oral vaccination with this live attenuated isolate protected fingerlings from E. ictaluri infection  induced cell-mediated immunity to protect catfish against ESC , which provided significant protection against ESC in both catfish fry and fingerlings  (gcvPΔsdhCΔfrdA) was constructed by in-frame deletion of three genes in the tricarboxylic acid cycle (sdhC and frdA) and one-carbon metabolism (gcvP)  . ESC-NDKE. ictaluri infection. Leukocytes containing E. ictaluri were detected in the AK of channel catfish at 48 h post-challenge (pc), and E. ictaluri was detected in the posterior kidney at 15 min pc  possesses hemopoietic tissue responsible for production of all blood elements –20. Seve5 min pc . In addinjection .E. ictaluri was detected in the abdominal area at 60–72 h pc  fish died in about 2 days, but at low doses (2.5 × 102-2.5 × 103 CFU) fish died in about 6 days. Dissection of catfish organs revealed that the AK and spleen had high E. ictaluri load, while E. ictaluri presence was also detected in the gills .The spleen in mammals is one of the secondary lymphoid organs in which antigen presentation occurs, and adaptive immune responses are activated . Similar–72 h pc . It was he gills . In the +) could promote CD4+ naïve T-cell stimulation (+ (L/CD207+) cells have been described in the AK and spleen of Atlantic salmon and rainbow trout. Also, the same research group has observed DC-like cells containing Birbeck -like granules in the gills of Chinook salmon during Loma salmonae infection , and described cells that resembled mammalian LC DCs containing Birbeck-like granules in the spleen, anterior and posterior kidneys, and gills of channel catfish by transmission electron microscopy (Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) and are critical players in bridging and shaping all innate and adaptive immune responses in vertebrates . Recentlmulation . Dendritmulation . Langerhmulation . Langerhmulation . Langerimulation , 37. Themulation . Severalnfection . Recentlcroscopy .It was demonstrated that the transcriptional regulator of the Zinc finger family DC-SCRIPT is expressed in all subsets of DCs in humans and mice , 42. DueE. ictaluri LAVs on innate and adaptive immune responses, and, specifically, on innate antigen presentation are still unexplored. Therefore, we aimed to evaluate the immune gene and L/CD207+ LC-like cells distribution patterns in the AK, spleen, and gills of catfish challenged with two LAV and WT strains of E. ictaluri.The effects of efficacious E. ictaluri strain 93–146, EiΔevpB, and ESC-NDKL1 were cultured in brain heart infusion (BHI) agar or broth  at 30°C. When required, media were supplemented with colistin .Wild-type (WT) EiΔevpB and ESC-NDKL1, E. ictaluri WT (positive control), and BHI (sham-control) as previously described  and placed in 10% neutral buffered formalin for 48 h . For immunohistochemistry (IHC) analysis, anterior kidney (AK), spleen, and gill tissues were isolated, dehydrated with a graded series of ethanol, and embedded in paraffin wax. Finally, tissue sections were cut as described previously .Two hundred specific-pathogen-free (SPF) channel catfish fingerlings (6-month old) were obtained from the College of Veterinary Medicine's fish hatchery at Mississippi State University, and all fish experiments were conducted according to the protocol approved by the Institutional Animal Care and Use Committee. Catfish were stocked into four 40-L tanks (25 fingerlings per tank) supplied with flow-through water and continuous aeration. Catfish were maintained at 25–28°C and fed twice daily with a floating catfish feed. After a week of acclimation, catfish were exposed to vaccine strains escribed . Exposureviously . For gen+ cells during E. ictaluri infection, immunohistochemical staining of catfish AK, spleen, and gill tissue was performed with polyclonal antibodies (pAbs) specific to human CD207  as described previously   . The L/CTo isolate total RNA from the tissues, the FastRNA™ SPIN Kit for Microbes and the FastPrep-24™Instrument  were used according to the manufacturer's instructions. DNase treatment with RNase-Free DNase Set  was used to eliminate any catfish gDNA contamination. The quantity and quality of total RNA were checked by using a NanoDrop ND-1000 spectrophotometer  and agarose gel.We used Maxima First Strand cDNA Synthesis Kit for RT-qPCR  to convert total RNAs into cDNA according to the manufacturer's instructions. The cDNA was synthesized in a 20 μl reaction containing 2.5 ng of total RNA, 4 μl of 5X reaction mix, 2 μl of maximum enzyme mix, and nuclease-free water. The reactions were incubated at 25°C for 10 min, then at 50°C for 15–30 min and at 85°C for 5 min. After that, reactions were stored at −80°C.http://bioinfo.ut.ee/primer3-0.4.0/), and the 3′ end of one primer was placed on intron/exon junction to eliminate any potential non-cDNA amplifications. Primers were synthesized commercially (MWG Eurofins Genomics), and real-time PCR was run using FastStart Universal SYBR Green Master Kit . Each qPCR reaction was 20 μl and included 10 μl FastStart Universal SYBR Green Master (ROX), 0.6 μl primers, 6.8 μl nuclease-free water, and 2 μl of cDNA. The 7,500 Real-Time PCR System (Applied Biosystems) was used to perform qPCR reactions for this study. Thermal cycler was programmed with 45 cycles of 95°C for 10 s, 95°C for 15 s, 57°C for 30 s, and 72°C for 15 s. Each sample was run triplicate.Immune genes and primers used in this study are listed in −ΔΔCt, and used for statistical analysis to determine significant differences between treatments EiΔevpB, ESC-NDKL1, and E. ictaluri WT.For normalization, the cycle threshold (Ct) of the 18 rRNA gene was subtracted from the Ct value of the gene of interest as described in the formula: ΔCt = Ct (target gene)—Ct (reference gene) . Then, t+ cells. The level of significance for all tests was set at P < 0.05.One-way and two-way ANOVA procedures of SAS  were used to evaluate differences in means of L/CD207EiΔevpB and WT strains, followed by rapid declines at 1 and 3 d, then partial recovery after 7 d post-exposure to the ESC-NDKL1 strain  and lipopolysaccharide (LPS), but relative expression of DC-SCRIPT was low at 3 and 7 d (The ESC-NDKL1-induced IFN-γ gene expression patterns in general resembled the patterns induced by the rophages , 16. In oth LAVs , Ibrahim in fish , 16. In  and 7 d .E. ictaluri WT. Interestingly, catfish fingerlings that survived the WT strain challenge 7 d pc showed significantly increased multiple adaptive immunity-related genes such as CD4 and CD8 T cell co-receptors and MHC class II, suggesting initiation of antibacterial immune responses under inflammatory conditions. Significantly elevated ESC-NDKL1-induced TNF-α gene expression levels in the spleen of catfish at 7 d pc could explain the higher mortality rates in catfish fingerlings challenged with the ESC-NDKL1 strain compared to its LAV counterpart (Importantly, significant increases in the expression of Th1 type polarization-related genes of adaptive immune responses were documented in the spleen at 7 d post-treatment with ESC-NDKL1 only. Namely, the IFN-γ gene was upregulated after exposure to the ESC-NDKL1 strain at 7 d post-exposure. Also, significant increases in the gene expression of T cell co-receptors, CD4-2 and CD8-β were evident in the spleen at 7 d post-exposure to the ESC-NDKL1 strain. Although as bacterial loads decreased , the domnterpart .+ cells in the spleen at 6 h pc, possibly via cytokine signaling cascades involving DCs, are due to the two LAVs being more efficient at inducing innate immune responses compared to their WT counterpart. The significant drops in the numbers of L/CD207+ cells in the spleen at 3 d pc suggest possible migration of these cells from the spleen to the site of infection followed by remarkable increases in LC-like numbers in the spleens of the vaccinated catfish after 7 d of the treatment. The LAVs induced increases in the spleen but not in the AK after 7 days of challenge, suggesting the migration of LC-like cells back to the spleen to present the pathogen-derived antigen to specific T cells, thus initiating adaptive immune responses. Similarly to our findings, the study with barramundi reported that the expression of DC-SCRIPT was higher in the spleen at 6 h post-injection of LPS and PTG, but its expression level decreased in the spleen after 1 d, and DC-SCRIPT expression was again elevated at 7 d (+ cell numbers. However, in the spleen, their significant increases were evident at 3 and 7 d compared to their counterparts at 6 h and 1 d post-treatment with both LAVs. The MHC class II molecules, unlike the DC-SCRIPT assessed in barramundi, are not limited to the pan-DC populations only but also expressed in other professional APCs, B cells and monocytes/macrophages (Our data on dramatic increases in the numbers of L/CD207d at 7 d . The MHCrophages –52.E. ictaluri into the channel catfish host (+ cells in the gills in all fish at 6 h, 1 and 3 d pc followed by decreases after 7 days could be interpreted as possible antigen recognition and capture by immature DCs. Our data support the previous observation that DC-like cells have been described in the gills of Chinook salmon heavily infected with L. salmonae (+ cells expression patterns in catfish gills at different time points of exposure to the LAVs and WT E. ictaluri strains. Our data suggest that L/CD207+ cells in the gills may recognize and capture antigens at 6 h, 1 d, and 3 d pc, and after 7 d, L/CD207+ cells in the gills of catfish vaccinated with two LAV strains have migrated to the secondary lymphoid tissue in the spleen for antigen presentation. However, the APC function of L/CD207+ cells and, in particular, their migration to the spleen in the gills of catfish infected with WT strain was impaired.Gills are one of the potential routes of entry for ish host . Our datsalmonae . We expaIn conclusion, followed by our previous observations on the vaccine strain-dependent differences in bacterial tissue persistence, fish mortality rates, humoral immune responses, uptake, and bacterial killing properties of macrophages , 16 hereLP and AK conceived and designed the experiments. LP and AK provided the original idea of the study. AOK, HoA, and HaA performed the experiments. LP, AK, and WB contributed reagents, materials, tools. AOK wrote the first draft of the manuscript and was involved in all aspects of the study. All authors were involved in critical interpretation of the data, manuscript revision, and final version approval.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli K-12, which raises the question of their role(s) in receptor function. Here we have investigated the roles of two polyP motifs in the osmosensor and histidine kinase EnvZ. We show that the IPPPL motif in the HAMP domain is required for dimerization of EnvZ. Moreover, replacement of the prolines in this motif by alanines disables the receptor’s sensor function. The second motif, VVPPA, which is located in the periplasmic domain, was found to be required for interaction with the modulator protein MzrA. Our study also reveals that polyP-dependent stalling has little effect on EnvZ levels. Hence, both polyP motifs in EnvZ are primarily involved in protein-protein interaction. Furthermore, while the first motif occurs in almost all EnvZ homologues, the second motif is only found in species that have MzrA, indicating co-evolution of the two proteins.Although distinct amino acid motifs containing consecutive prolines (polyP) cause ribosome stalling, which necessitates recruitment of the translation elongation factor P (EF-P), they occur strikingly often in bacterial proteomes. For example, polyP motifs are found in more than half of all histidine kinases in  The osmolality of these media was measured with an osmometer . For selection purposes, antibiotics were added at concentrations of 50 μg/ml (kanamycin) and 100 μg/ml (ampicillin).envZ of the E. coli strains MG1655 rpsL150 . All subsequent preparation steps were performed at 4°C. After high-pressure cell disruption, cell debris was removed by centrifugation as described above. Membrane vesicles were then prepared by ultracentrifugation of the supernatant at 250,000 x g for 45 min. The proteins of the cytoplasmic membrane were solubilized by resuspension of the pellet in 2 ml of Na-phosphate buffer  containing 2% (w/v) Triton X-100 and incubation at 37°C for 1 h. The suspension was then centrifuged at 390,000 x g for 30 min. The supernatant was discarded, and the pellet was washed in Na-phosphate buffer without Triton and centrifuged again at 390,000 x g for 30 min. The final pellet was resuspended in 100 μl PBS  and SDS sample buffer was added . Samples were then supplemented with 25 mg urea and heated at 100°C for 5 min before gel electrophoresis (5 μl per lane).Proteins were fractionated on a 12.5% (w/v) SDS polyacrylamide gel (acrylamide:bis-acrylamide 37.5:1). Concentrated outer membrane proteins were fractionated on a 10% SDS polyacrylamide gel (acrylamide:bisacrylamide 44:1) supplemented with 4 M urea . Gels we2CO3/NaHCO3 pH 9.5), the membrane was scanned and the bands quantitatively analyzed using the software ImageJ.Proteins were transferred onto PVDF membranes by wet blotting. EnvZ was detected with specific anti-EnvZ antibodies , or anti-FLAG antibodies (abcam) diluted 1:5,000 in TBS-T buffer  supplemented with 0.75% (w/v) skim-milk powder. Bound antibodies were detected with alkaline-phosphatase-conjugated anti-rabbit or anti-mouse antibodies (Rockland), diluted to 1:4,000 in TBS-T buffer. After developing the blot with NBT/BCIP  were grown overnight in M9 medium. This culture was then used to inoculate fresh M9 medium, and cells were grown to mid-exponential phase. Cultures were diluted 1:500 in fresh M9 medium supplemented with NaCl or sucrose depending on the experiment. Aliquots (200 μl) of each culture were transferred to Greiner 96-well plates and incubated under constant agitation at 37°C to an OD600 of about 0.2. After a 15-min incubation on ice, fluorescence was measured with a TECAN Reader .This assay is based on the previously published method . ReportemzrA or envZ coding sequence was fused to the 5’ end of pKT25 or pT18 (Euromedex) encoding the corresponding, complementing adenylate cyclase fragments [envZ: the wild-type gene, and envZ with codon substitutions that converted the VVPPL motif into VVAAL, or IPPPL into IAAAL, or both replacements together.The ragments , 48. We E. coli reporter strain BTH101 [600 of 0.2 were plated on LB agar supplemented with 1 mM IPTG and 40 μg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside . After incubation at 30°C for 24 h, galactosidase activity was quantified based on the intensity of the blue color of the colonies.For the analysis of protein-protein interactions, we used the n BTH101 , which wn BTH101 . In paraE. coli K-12 was downloaded from the UniProt database (UniProt Entry: P0AEJ4) and used as the template for modelling of its 3-D structure with Phyre 2.0 [The full-length EnvZ sequence from hyre 2.0 . For thehyre 2.0 .E. coli K-12 as the query sequence . Sequences shorter than 90% of the E. coli K-12 EnvZ sequence were excluded. A pairwise alignment of 793 sequences was done with a progressive algorithm from the software CLC Workbench 7.6 , using the following parameters: gap open cost 10, gap extension cost 1, high accuracy. The results served as the basis for construction of a phylogenetic tree by the software’s high-accuracy, distance-based neighbor-joining algorithm (100 bootstrap replicates and the Jukes-Cantor distance correction as default parameters). We screened these organisms for MzrA by searching for orthologues of E. coli K-12 MzrA with NCBI Protein BLAST .To identify non-redundant EnvZ orthologues, we carried out a BLAST search of the UniProt Microbial Proteomes database using the full-length EnvZ from E. coli K-12 EnvZ and MzrA orthologues  from the Gammaproteobacteria included in the tree of life recently published by Hug et al. [In addition, we selected both g et al. . The treg et al. , 54. To E. coli EnvZ contains an IPPPL motif in the HAMP domain (polyPc), which is thought to cause strong translational arrest [E. coli K-12 EnvZ. Among the Gammaproteobacteria, we found 63 species that have EnvZ (proteins that show 44–100% sequence identity to the EnvZ from E. coli K-12). Sequence alignment revealed strong conservation specifically of the prolines in the IPPPL motif: Ile201 60%, Pro202 89%, Pro203 68%, Pro204 81%, Leu205 87%  assay, which is based on the split adenylate cyclase (CyaA) from Bordetella pertussis [c motif were replaced by alanines. The assay is based on the (dimerization-dependent) complementation of the adenylate cyclase fragments (T25 and T18), which are translationally fused to the cytoplasmic N-terminal ends of EnvZ. Functional reassembly of the adenylate cyclase induces a cAMP signalling cascade, which activates transcription of the lac operon in the E. coli reporter strain BTH101 [HAMP domains form a homodimeric, four-helical, parallel coiled-coil structure and are crucial for signal transduction of receptor proteins –58, but ertussis , 60. We n BTH101 . As a poompC and ompF via OmpR.To further investigate the importance of the IPPPL motif for the signaling activity of EnvZ, we replaced it with IAAAL in the chromosomally encoded EnvZ and characterized the mutant’s response to osmotic stress by measuring in two distinct ways the ability of EnvZ to regulate the expression of E. coli MG1655, and the isogenic mutants ΔenvZ and EnvZP/A(c) (replacement of IPPPL by IAAAL) were grown in media of increasing osmolarity imposed by the addition of NaCl. Outer membrane proteins were isolated and visualized on a Coomassie Blue-stained SDS-urea gel  variant was associated with high OmpC levels and extremely low OmpF levels under non-stress conditions. In response to osmotic stress only a slight increase in OmpC and decrease in OmpF were observed. This suggests that the EnvZ variant is locked in an ON state [In the first approach, wild-type urea gel . In addiurea gel . In wildON state .ompC-cfp fusion in the reporter strain EPB273a [envZ mutant produced very low levels of OmpC-CFP protein and responded weakly to osmotic stress. The EnvZP/A(c) mutant was characterized by significantly higher OmpC-CFP levels in comparison to the wild type under all tested conditions  in the periplasmic domain. This motif is one of the medium-to-weak stalling motifs . The perla75 60% .p motif is involved in the interaction of EnvZ with the membrane-integrated MzrA, the modulator protein of EnvZ. Previously, it was shown that two amino acid substitutions in the periplasmic domain of MzrA are sufficient to decrease significantly its capacity to bind to EnvZ [Because of its exposed localization within the periplasm, we asked whether the polyP to EnvZ . Therefop motif led to a decline in the β-galactosidase activities to about 60%, suggesting a decreased affinity of this EnvZ variant for MzrA. In contrast to the polyPc motif, the polyPp motif does not contribute to the dimerization of EnvZ  . The color code illustrates the presence of polyPp and polyPc in EnvZ as well as the occurrence of a MzrA homologue in the respective organisms  is widely distributed. This underlines the importance of the triple proline in the HAMP domain and its higher degree of conservation  . By NCBIrganisms . The polervation . In contE. coli K-12 EnvZ by BLAST homology search and marked them in the tree according to the percentage of sequence identity. In parallel, we screened for the presence of E. coli K-12 MzrA orthologues (> 31% sequence identity) and depicted the presence of polyP motifs in EnvZ with a color scheme.To trace the emergence of the two polyP motifs during evolution, we made use of the above- mentioned phylogenetic species tree of Gammaproteobacteria  Fig 5B)Fig 5B). p seems to have evolved together with MzrA. All organisms that encode MzrA also have a polyPp motif in their EnvZ homologues. In addition, we analyzed the development of the putative stalling strength of the proline motifs. Again, we referred to Qi et al. for motif classification [Budvicia, Pragia, Salinivibrio, Photobacterium, Vibrio, Alteromonas, Algicola and Rheinheimera. In EnvZ both motifs seem to be a consequence of a specialization, in contrast to the expectation that polyP motifs in general developed early during evolution and constantly underlie selection pressure [Both proline motifs arose late in evolution, and polyPfication . There ipressure , 24.E. coli K-12. Both motifs are located in exposed structural regions in EnvZ, and we found that they are important for protein-protein interactions. First, we focused on the IPPPL motif, which is thought to induce a strong translation arrest [envZ transcripts was artificially increased by cloning the gene into a plasmid under the control of the PBAD promoter  variant is shifted strongly towards a kinase ON state  variant.MzrA is known to interact with EnvZ in the periplasm . Unexpecytoplasm , which wand MzrA  and a reand MzrA . Therefop motif for the interaction with the modulator MzrA is supported by a phylogenetic analysis, which revealed co-evolution between MzrA and polyPp Click here for additional data file.


M. longissimus injected with thermolysin (80 ppm) and stored for 3 days at 5°C.This study identified angiotensin I-converting enzyme (ACE) inhibitory peptides in beef 50) of each peptide was evaluated.Crude peptides (molecular weight <3 kDa) were obtained from the thermolysin hydrolysate and separated into seven fractions. Fraction V showing the highest ACE inhibitory activity was further fractionated, yielding subfractions V-15, V-m1, and V-m2, and selected for superior ACE inhibitory activity. Finally, twelve peptides were identified from the three peak fractions and the ACE inhibitory activity  and had higher concentrations  relative to the other peptides tested.The Leu-Ser-Trp, Phe-Gly-Tyr, and Tyr-Arg-Gln peptides exhibited the strongest ACE inhibitory activity (ICThese results suggest that the thermolysin injection process is beneficial to the generation of bioactive peptides with strong ACE inhibitory activity. The system starts with the conversion of angiotensinogen to a prehypertensive hormone, angiotensin-I , by the action of renin secreted by the kidney . Angiotephalitis . The RASphalitis .Usually, ACE inhibitors are isolated naturally in mammals within the gastrointestinal tract during the normal metabolism of proteins . HoweverM. longissimus from beef generated by the injection of thermolysin followed by aging for 3 days at 5°C and to evaluate the ACE inhibitory activity of the identified peptides.The brine injection or marination process is commercially used for the quality enhancement of fresh meat . VariousThermolysin , ACE (from rabbit lung), hippuryl-L-histidyl-L-leucine (HHL), hydrindantin, and lithium hydroxide were purchased from Sigma (USA). Acetonitrile (LC-MS grade) was purchased from Roth GmbH ; L-Leucine from Aldrich Co. ; dimethyl sulfoxide (DMSO) from Merck ; ninhydrin from Riedel-de Haen ; and acetic acid and sodium hydroxide from Hayashi Pure Chemical Ltd. .M. longissimus) were obtained from Hanwoo steer  slaughtered at a commercial slaughterhouse. The beef loins (200 g) were injected with 80 ppm of thermolysin and stored for 3 days at 5°C for enzymatic proteolysis of the muscle proteins. The use of thermolysin and its concentration were determined based on our preliminary study. The process for isolation and identification of ACE inhibitory was presented in At 24 h postmortem, loins  to remove high molecular weight peptides. The filtrates were then centrifuged at 4,000 g or 45 min at 4°C using an Ultracel 3K membrane . Prior to use, the membrane was activated with 10 mL of distilled water, and the remaining liquid was carefully removed. Separated crude peptide extracts were subsequently lyophilized and stored at −80°C until required for ACE inhibitory activity analysis and further separation.The peptides were extracted from the prepared beef samples as described in Jang and Lee . BrieflyThe lyophilized crude peptides were dissolved in distilled water and again filtered through a Millipore membrane filter (0.45 μm) and applied to a column  saturated in P-2 resin. The eluent was deionized with distilled water with a flow rate of 0.8 mL/min. The fractions were collected at 15 min intervals with a fraction collector  and all fractions were subjected to absorbance measurements at 220 nm to confirm the peptide concentration. The fractions were collected on the basis of molecular weight and subsequently lyophilized prior to the ACE inhibitory activity measurements.The highest ACE inhibitory fraction selected after gel filtration was dissolved in water containing 1% trifluoroacetic acid (TFA). For the quantitative analysis of ACE inhibitory peptides, reversed-phase high-performance liquid chromatography (RP-HPLC) was performed with a Jasco PU-980 gradient system with a Jasco photodiode array (PDA) detector at 220 nm . The fraction solution (20 μL) was applied to a Vydac C18 reverse-phase column  and a solution containing 0.5% TFA, 0.5% acetonitrile (ACN), and 0.1% TFA was used for mobile phase at a flow rate of 1.0 mL/min. The seven major fractions were collected and subsequently lyophilized and the ACE inhibitory activity and concentration were measured. Among the seven fractions, the three fractions displaying strong ACE inhibition were applied to an ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) system to analyze the amino acid sequence of the peptides in the fractions.An UPLC system  equipped with a binary solvent delivery system, an autosampler, and a PDA detector was used to analyze the peptides. An Acquity UPLC BEH C18 column  was equilibrated with water containing 0.1% TFA. HPLC fractions (5 μL) were injected into the column and eluted with an ACN gradient containing 0.1% TFA at a flow rate of 0.35 mL/min for 12 min. The absorbance of the eluent was detected at 214 nm by the PDA detector and QTOF-MS/MS.The peptides from thermolysin hydrolysates of beef loin were separated and analyzed by UPLC-QTOF-MS/MS using a reverse phase C18 column . The Q-TOF-MS/MS  was operated in ESI positive mode. The capillary and sampling cone voltages were set at 2.78 kV and 26 V respectively. The desolvation flow was set to 700 L/h at a temperature of 300°C and the source temperature was set at 100°C. The QTOF MS data was collected in a 50 to 1,000 m/z range with a scan time of 0.2 s and an interscan delay time of 0.02 s. The MS/MS spectra of the peptides were obtained using a collision energy ramp from 10 to 45 eV. The accurate mass and composition of the precursor ions and fragment ions were calculated and sequenced using MassLynx software  and the peptides were analyzed and sequenced by a BioLynx peptide sequencer .g for 10 min at 4°C, and a 0.7 mL aliquot of each ethyl acetate layer was transferred to clean tubes and evaporated by heating at 95°C for 20 min in a water bath. The hippuric acid was redissolved in 3 mL of 1 M NaCl, and the amount formed was determined by absorbance at 228 nm. The IC50 value, defined as the concentration of a peptide that inhibits 50% of the ACE activity, was determined by measuring the ACE inhibitory activity and the peptide contents of each of the extracts after regression analysis. ACE inhibitory activity (%) was determined using the equation:ACE inhibitory activity determination was achieved using the spectrophotometric method described by Cushman and Cheung . For eacwhere S and B are optical density (OD) values for the sample and blank, respectively, and SC and BC are the OD values for the sample control and blank, respectively.The identified peptides were chemically synthesized by Peptron Ltd.  using the PenptrEX automatic peptide synthesizer and lyophilized for further confirmation of ACE inhibitory activity.The entire experiments were replicated three times individually and all the samples were analyzed in duplicate. All data were expressed as means±standard deviation. One-way analysis of variance was carried out to analyze the data using SAS program . The treatment (fraction or peptide type) was the fixed effect in the model. Comparison of mean values was performed to find out the effect of treatment using Duncan’s multiple range tests. A value of p<0.05 was considered significant differences.The thermolysin hydrolysates were filtered (MW <3 kDa) by ultrafiltration using PM-10 and Ultracel 3K membranes. After gel filtration, seven fractions were separated from the extract derived from beef loins injected with thermolysin and the ACE inhibitory activity of each fraction was evaluated at a concentration of 1 mg/mL. Fraction V inhibited ACE activity at the highest rate (66.5%), and the other five fractions  achieved 50% inhibitory activity at a concentration of 1 mg/mL . This reThe major peak fractions  were separated from fraction V by RP-HPLC. The most potent fractions  that had the higher peptide concentrations and ACE inhibitory activities  were sel+) and a total of twelve candidate peptides was identified from fraction V-15, V-m1, and V-m2 (50 = 0.89 mM) from fraction V-15. The quantitative analysis of the three peptides with the highest ACE inhibitory activity that was generated by thermolysin-injected beef loin followed by 3 days of aging at 5°C—Leu-Ser-Trp, Phe-Gly-Tyr, and Tyr-Arg-Gln—was determined by LC-MS . These peptides were not detected in the non-injected control sample (data not shown). Many studies have found ACE inhibitory peptides derived from red meat, poultry, and fish muscle using proteolytic enzymes or processing, including aging or fermentation [The total mass of the peptide mixture in each fraction was obtained from the mass detector, and the monoisotopic mass of each individual amino acid was subtracted to identify the exact sequence with an accurate mass. The MS/MS spectra of the potent peptides were displayed using a single positively charged ion  had an aromatic C-terminal Trp, corresponding with the results of another study [50 of 2.69 mM for ACE inhibitory activity, consists of an aromatic amino acid at the N-terminus and Tyr at the C-terminus. Tyr-Arg-Gln also consists of an aromatic amino acid at the N-terminus. In other words, the three amino acid-sequenced peptides  derived from thermolysin-injected beef with the greatest potential for use as ACE inhibitory peptides have highly similar sequence properties with peptides with well-established ACE inhibitory activity [There are many reported protein structure-function studies that use different protein sources. Previous research has demonstrated that ACE inhibitory peptides have some common characteristics: i) The C-terminal tripeptide sequence strongly affects binding to ACE even though the underlying mechanism has yet to be completely established ; ii) C-ter study . Additioer study ,25,26. Tactivity ,28. Fromactivity .in vivo.Toopcham et al  reportedin vivo will be a necessary step for their future use in the meat and meat products industry as functional biomaterials or a healthy dietetic method.In the present study, strong ACE inhibitory peptides—Leu-Ser-Trp, Phe-Gly-Tyr, and Tyr-Arg-Gln—were produced and identified from thermolysin-injected beef. Based on results through ultrafiltration, gel filtration, RP-HPLC, and UPLC-QTOF-MS/MS, injection of thermolysin into beef followed by 3 days refrigerated storage may increase ACE inhibition activity. Consequently, the consumption of the beef would be more beneficial for human health . Further


To evaluate the early growth (weight and length) of HIV-exposed uninfected (HEU) children from the prevention of mother-to-child transmission (PMTCT) of human immunodeficiency virus (HIV) program in Guangdong Province, China.A total of 731 HEU children were longitudinally followed up at 7 time points, with anthropometric measurement conducted of weight and length (supine) in the first 18 months. Z scores were calculated, with and without adjustment for gestational age.p < 0.05).A total of 708 HEU children were included in the final follow-up cohort, and 105 (14.83%) children completed all 7 follow-up visits. The mean of adjusted weight-for-age Z scores in these children was above zero and showed a decreasing trend in 18 months. The mean of adjusted length-for-age Z scores showed a decreasing trend and was above zero in the first 12 months; this declined to under zero at age 18 months. The proportion of underweight was 0.28–2.19% and that of stunting was 0.71–4.63% at each follow-up month-age. Slower growth in HEU children was associated with no sustained food subside after 6 month, mothers’ hemoglobin content less than 100 g/L during pregnancy, preterm birth, and low birth weight (HEU children could catch up to WHO growth standards in first 18 months in Guangdong; however, growth declined after 12 months, and these children need sustained nutritional support. With increased access to antiretroviral drugs (ARVs) for prevention of mother-to-child transmission (PMTCT) of human immunodeficiency virus (HIV) in China, most children born to HIV-infected mothers are not themselves infected with HIV , 2. ManySince 2011 in Guangdong Province of China, free ARV prophylaxis for PMTCT has been routinely provided to all HIV-infected pregnant women from 14 gestational weeks to postpartum, as well as to their newborns in the first 4–6 weeks after birth. The PMTCT program has effectively reduced the HIV mother-to-child transmission rate to less than 5% in Guangdong [The present longitudinal cohort study evaluating the growth of HIV-uninfected children born to HIV-infected mothers  was conducted in Guangdong Province, China from January 2012 to December 2016. A total of 731 HEU children included in the free HIV PMTCT program were recruited at birth in the hospital from January 2012 to June 2015. After written informed consent was obtained from parents or guardians, the children were followed up from birth to age 18 months, with anthropometric measures taken of weight and length (supine). We excluded the data of HEU children who were twins or multiple births, those who were HIV-infected, and those who died within the first 18 months. Finally, 708 eligible children were included in the original cohort and 105 children were followed up at all 7 time points.HEU children were followed up by local child health care doctors and nurses at birth in a delivery hospital, and they were followed up in a child health care service hospital at ages 1, 3, 6, 9, 12, and 18 months. Information of social demographic status, prenatal complications , CD4+ T cell level, and ARVs use by the mother during pregnancy were derived from the perinatal medical records and routine case report cards during the period of delivery in the hospital. At each time point of monthly planned visits, children underwent follow-up visits, including measurement of weight and length (supine) using routine calibrated instruments, physical examination, feeding consultation, and recording of the feeding status or any hospitalization for illness in children during the interval since the last follow-up visit. At the same time, children were referred for free services of the government PMTCT program, including HIV testing, provision of ARVs (azidothymidine or nevirapine) for HIV PMTCT in the child’s first 4 to 6 weeks of life, and provision of 3000 RMB to purchase infant formula milk in the first 6 months after birth.The HIV status of children was confirmed either by western blot analysis after seropositive tests results for HIV antibodies at ages 12 and 18 months or by HIV DNA polymerase chain reaction (PCR) tests using dried blood spot (DBS) specimens at ages 6 weeks to 3 months. Western blot analysis was conducted at the local city-level laboratory of the Chinese Center for Disease Control and Prevention using venous blood specimens; DBS testing was conducted in the Administrative Authorized HIV-testing laboratory of Guangdong Province Hospital for Women and Children Healthcare.Statistical analysis was conducted using R software version 3.5.1 (2018-07-02). We compared the difference of characteristics between all 708 children who were ever followed up and 105 children who were followed up at all 7 time points by a chi-square test. According to the World Health Organization (WHO) Child Growth Standards in 2006 , we calcp-value < 0.05.Underweight was defined as weight-for-age Z scores less than − 2, overweight as weight-for-age Z scores more than 2, and stunting as length-for-age Z scores less than − 2. The mean and 95% confidence interval (CI) and median of Z scores of weight for age, gestational age-adjusted weight for age, length for age, and gestational age-adjusted length for age were calculated and trend lines were plotted. Generalized linear mixed models (GLMM) were used to evaluate the effect of covariates on growth over time, and the age month was assigned to be a group variable, and other characteristics variables was assigned to be the covariates. Results were considered statistically significant with A total of 731 newborns born to HIV-infected mothers were included in the original follow-up cohort at birth. During the follow-up period of 18 months, 23 children were excluded from the original cohort because 6 children died, 16 children were HIV-infected, and 1 child died as a result of HIV infection. Finally, 708 HEU children were included in the cohort. Among 708 HEU children, 105 (14.83%) children underwent all 7 follow-up visits (including the first visit at birth); 133 (18.79%) children completed 6 visits; 102 (14.41%) children attended 5 visits; 87 (12.29%) children underwent 4 visits; 96 (13.56%) children attended 3 visits; 124 (17.51%) children completed 2 visits; and 61 (8.62%) children underwent 1 follow-up visit. The flow of the follow-up cohort of children is shown in Fig. P < 0.001) and 32.49%  of all 708 children.Characteristics of children and their HIV-infected mothers are shown in Table The trend of the mean and 95% CI and median of weight-for-age Z scores and that of gestational-age-adjusted weight-for-age Z scores of HEU children are shown in Figs. p < 0.05) are shown in Table The gestational age-adjusted weight-for-age Z scores of 105 seven-time followed-up HEU children who had characteristics with significant differences , at 1 month was , at 3 month was , at 6 month was , at 9 month was , at 12 month was , and at 18 month was .The Z scores of weight for age among the seven-time followed-up children compared with that among less than seven-time follow-up children, at birth was  was 0.28% (2/708), 0.67% (3/447), 2.19% (10/457), 2.01% (8/398), 1.51% (5/331), 0.82% (3/366), and 1.78% (5/281); and that of overweight  was 20.90% (148/708), 40.72% (182/447), 19.91% (91/457), 8.29% (33/398), 7.55% (25/331),8.20% (30/366), and 6.05% (17/281), respectively. In addition, there was a higher proportion of underweight after age 1 month.The trend of the mean and 95% CI and median of length-for-age Z scores and that of gestational age-adjusted length-for-age Z scores of HEU children are shown in Figs. p < 0.05) are shown in Table The gestational age-adjusted length-for-age Z scores in 105 seven-time followed-up HEU children with characteristics that showed significant differences , at 1 month was , at 3 month was , at 6 month was , at 9 month was , at 12 month was , and at 18 month was .The Z scores of length for age among the seven-time followed-up children compared with that among less than seven-time follow-up children, at birth was , 1.57%(7/447), 4.60%(21/457), 4.02%(16/398), 4.53%(15/331), 2.73%(10/366), and 4.63%(13/281) respectively.A previous review report has shown that HIV prevalence among pregnant women in China who access antenatal care remains below 0.1% . HoweverFeeding is one of main factors influencing the growth of infants. In China, formula feeding consultation and a subsidy of 3000 RMB for formula milk are routinely provided for all children born to HIV-infected mothers. Our results showed that there was a significantly higher proportion of formula-fed infants at 3 months (94.29% vs. 63.68%) and 6 months (94.29% vs. 57.79%) among the 105 HEU children followed up at all 7 time points compared with the 708 ever followed-up HEU children. However, there were no differences in growth between the different feeding models in both two groups of children. In this study, few HEU children were exclusively breast fed. These results might indicate that artificial feeding, including formula feeding for HEU infants in Guangdong in the first 6 months is suitable for their growth of weight and length; however, feeding consultation and support for these children should be reinforced after 6 months. Previous studies have found that early growth in HEU children is lower than that in HUU children, partly owing to mastitis with breastfeeding that cannot be resolved with intensive support for exclusive breastfeeding . InterveThe results of GLMM also showed that preterm and low birth weight might are both the influencing factors on growth among HEU children in the first 3 month, but a potential long-term effect of preterm and low birth weight on the growth of these children need further study as the cases were not enough in our study. We also found that mothers’ hemoglobin content less than 100 g/L during pregnancy was associated with lower gestational age-adjusted weight-for-age Z scores in the first month. These results suggest that the growth of HEU children should continue to be intervened from pregnancy throughout childhood in this population. Many previous reports have shown that the health and development problems of HEU infants are multi-factorial , 9, and In this study, we used the WHO Child Growth Standards in 2006 but we used a growth reference for China to calculate Z scores and adjusted Z scores of weight for age and length for age. Previous studies have shown that the WHO growth standards might be more conservative in estimating undernutrition and are more applicable for international comparisons with Chinese children . There iOne limitation of this study is the large loss to follow-up over the seven time points, which was owing to a change in the method of contact and patient relocation. But we found no significant differences between the group of ever followed-up children and the group who completed all seven follow-up visits, except for infant formula feeding in the first 3 months and the first 6 months, which could mitigate the biases of loss to follow-up. Another limitation of this study was that we had no control follow-up group; however, the purpose of this study was to evaluate the growth status of weight and length among HEU children, which can be accomplished by comparison with the WHO growth standards. And our results also showed that HEU children had a similar growth pattern as general children in China, and the pattern is the decreasing prevalence of low weight and growth stunt and increasing prevalence of overweight and obesity of children , 32.In conclusion, HEU children from the PMTCT of HIV program could catch up to WHO growth standards in first 18 months in Guangdong; however, growth declined after 12 months, and these children need sustained nutritional support. Growth in terms of weight and length in HEU children is associated with, mothers’ hemoglobin content less than 100 g/L during pregnancy, preterm birth, and low birth weight. Findings also highlight the necessary to provide continued professional support and intervention to improve growth in HEU children, from pregnancy throughout their entire childhood.


Micrococcaceae and Clostridiaceae-1 were potentially correlated to the severity of allergic airway inflammation. Our study suggests that perinatal and early-life vancomycin intervention aggravates allergic inflammation in adulthood, which might be correlated with imbalanced gut and lung microbiome homeostasis.Several studies have reported that gut and lung microbiomes are involved in the process of asthma pathogenesis. However, it remains unclear how perinatal or early-life antibiotic intervention affect adult allergic airway inflammation. We assigned C57BL/6 mice randomly to four experimental groups: normal saline control (NS), ovalbumin (OVA), vancomycin pretreated NS (VAN-NS), and vancomycin pretreated OVA (VAN-OVA). The vancomycin groups were orally given the drug from gestational day 14 to 6 week. An OVA-induced asthma model was then established at 6 weeks of age, and airway inflammation was evaluated. In addition, total DNA was extracted from the feces and lung tissue and used for 16S rDNA gene sequencing, to detect the composition of the microbiome. In the VAN-OVA group, airway inflammation and Th2-related cytokines were found to be significantly increased versus the control groups. Gene sequencing showed that vancomycin treatment attenuated the richness and evenness, and altered the composition of the microbiome in the gut and lung.  Asthma is defined as an allergen-induced chronic airway inflammation disease, characteristic of airway hyperresponsiveness (AHR), reversible airflow restriction, and airway remodeling. The clinical symptoms include coughing, wheezing, dyspnea, and chest tightness. Asthma is thought to be caused by a combination of genetic and environmental factors . More reThe last century has shown an alarming rise in immune dysregulation diseases, leading to the hygiene hypothesis. The prevalence of these immune disorders is thought to be associated with reduced type 1 immune responses. In the past few decades, several studies have demonstrated that microbial dysbiosis is important for immune imbalance—especially the microbial colonization and immunostimulatory signals during early life or those passed on by the mother .in utero; gut microbiota colonization occurs after birth [Firmicutes and Bacteroidetes in the intestinal microbiome of healthy humans; there are also some Proteobacteria, Actinobacteria, Verrucomicrobia, and Cyanobacteria [The occurrence and development of infectious diseases, obesity, diabetes, liver diseases, coronary heart disease, tumors, and hypersensitivity disease are closely related to the microbiome dysbiosis –11. Manyer birth . In the er birth . The dombacteria ,16.It has also been reported that neonatal and continuous vancomycin administration in the murine allergic asthma model increases major indicators of airway inflammation . In contTo clarify the effect of antibiotics in early life on asthma, we established the following models: mice being exposed to vancomycin from late pregnancy  to 6 weeks of age, when an OVA-induced asthma model was established . CompareWe then examined the pulmonary pathology sections stained with hematoxylin/eosin (H&E) and periodic acid-Schiff (PAS) staining. In the OVA-induced asthmatic mice, inflammatory cells around the trachea and mucin secretion markedly increased in comparison to the control group. In the vancomycin-treated group, both the inflammation cells and mucus production were significantly increased versus the OVA group .To further investigate how vancomycin affects OVA-induced airway inflammation, we collected the feces and lung tissue of the mice, and examined the composition of the microbiome via 16S rDNA gene sequencing. The differences in the species and quantity of microbiota were obvious between these groups.The rank-abundance curve describes the richness and evenness of the microbial species and showed that these difference between groups were insignificant. In the VAN-NS and VAN-OVA groups, the richness and evenness of the microbiota was reduced in the gut and the lung, compared to the NS and OVA groups . This suBacteroidia , Clostridia , and Betaproteobacteria . In the feces of OVA-induced allergic asthma mice, the abundance of Clostridia distinctly decreased to 15.48%, and the Bacteroidia increased to 74.35%. In the VAN-OVA group, Clostridia decreased from 15.48% to 8.14%, while Bacteroidia was enriched to 82.45% from 74.35%; Betaproteobacteria was enriched to 5.3% from 1.9%, versus the OVA group , Bacilli , and Alphaproteobacteria . In the OVA group, the proportion of Bacilli decreased from 16.79% to 3.6%, while Alphaproteobacteria decreased from 14.81% to 5.6% compared to the NS group. This tendency to decrease was heightened with vancomycin treatment. In the VAN-OVA group, the proportion of Bacilli decreased to 1.88%, and Alphaproteobacteria was 4.42% in the lung microbiome. There are many bacterial genera that increased with vancomycin intervention: Betaproteobacteria was enriched to 59.86%, while the proportion was 33.44% in the OVA-induced asthma group  were purchased from the Slac Laboratory Animal Co. Ltd. . Breeders were mating them, and their offspring were selected for the following experiments: all mice were maintained in a specific-pathogen-free facility and given a standard diet with normal drinking water. The room temperature was maintained at 23 ± 2° C with 50% ± 10% humidity and a 12-h light/12-h dark cycle. The study was conducted in agreement with the Experimental Animal Welfare and Ethics Committee of Zhejiang University.C57BL/6 pregnant mice were randomly assigned to normal drinking water, with vancomycin-treatment groups assigned on day 14 of gestation. Mice in the vancomycin-treatment group were given vancomycin at 200 mg/L in drinking water from gestational day 14 to 6-weeks old. The offspring of control and vancomycin-treatment mice were randomly divided into normal saline  and OVA (n = 10) groups, respectively. Mice in the non-vancomycin-treatment group had drinking water for the entire experiment.The OVA-induced asthma model was established in mice at 6-weeks old. Mice in the OVA and VAN-OVA groups for the asthma model were sensitized on day 0 and 14 by intraperitoneal injection of 80 ug OVA  in 0.1 ml and an equal amount of aluminum hydroxide . The NS groups were given NS instead. On days 24 to 26, mice were challenged with 1.5% OVA for 35 min. Control mice in the NS and VAN-NS groups were exposed to saline aerosols. Parameters were analyzed at 24 h after the OVA challenge.At 24 h after final OVA challenge, mice were anesthetized and sacrificed for airway inflammation analysis. BALF was extracted from the left lungs with 0.4 ml PBS three times for a total volume of 1 ml. Total BALF cells were counted under a microscope. The remaining cells were centrifuged, spun onto glass slides, and stained with Wright-Giemsa staining buffer according to the manufacturer’s instructions; the number of differential cells were counted and classified under a microscope by finding > 200 cells.The left lungs were fixed in formalin for 24 h and then embedded in paraffin. The sections were stained with H&E and PAS. The inflammation was evaluated as described with the H&E staining sections . The PASIl-13: forward: 5′-CAGCCTCCCCGATACCAAAAT-3′, reverse: 5′-GCGAAACAGTTGCTTTGTGTAG-3′; Il-4: forward: 5’-CCCCAGCTAGTTGTCATCCTG-3’, reverse: 5’- CAAGTGATTTTTGTCGCATCCG-3’; β-actin: forward: 5′-GTCCACCGTGTATGCCTTCT-3′, reverse: 5′-CTCCTGGTGTCCGAACTGAT-3′.The lung tissue was lysed with Trizol reagent . The cDNA was amplified with Reverse Transcription Reagents (Takara Biotechnology). The expression of IL13 and IL4 was measured by Q-PCR, using SYBR Green Master Mix  with a StepOne real-time PCR system . Primers used IL13 and IL4 Q-PCR as follows: The feces and lung tissue were cut in pieces with sterile scissors, and total DNA was extracted with QIAamp DNA Mini Kit  according to manufacturer’s instructions. The DNA quality and concentration (purity ratio A260/A280) were measured with a NanoDrop ND-1000 spectrophotometer, and stored at -80° C for other experiments.The 16S rDNA genes of the V4 regions were amplified, using specific primer  with the barcode. All PCR reactions used Phusion® High-Fidelity PCR Master Mix . We mixed the same volume of 1X loading buffer (which contained SYB green) with PCR products and performed electrophoresis on 2% agarose gel for detection. Samples with a bright main strip between 400-450 bp were chosen for further experiments. The PCR products were mixed in equal density ratios. A mixture of PCR products was then purified with Qiagen Gel Extraction Kit . Sequencing libraries were generated using TruSeq® DNA PCR-Free Sample Preparation Kit  following the manufacturer's recommendations with index codes added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Fisher Scientific) and the Agilent Bioanalyzer 2100 system . Finally, the library was sequenced on an IlluminaHiSeq2500 platform and 250 bp paired-end reads were generated.The statistical differences among groups were analyzed with one-way ANOVA, and P values were shown in related graphs. All data were expressed as the mean ± SEM. The analyses and graphs were performed with GraphPad Prism 7.0 software . The level of statistical significance was set at a p-value < 0.05.Supplementary Figure


Selecting a target based on a representation in visual working memory (VWM) affords biasing covert attention towards objects with memory-matching features. Recently, we showed that even task-irrelevant features of a VWM template bias attention. Specifically, when participants had to saccade to a cued shape, distractors sharing the cue’s search-irrelevant color captured the eyes. While a saccade always aims at one target location, multiple locations can be attended covertly. Here, we investigated whether covert attention is captured similarly as the eyes. In our partial report task, each trial started with a shape-defined search cue, followed by a fixation cross. Next, two colored shapes, each including a letter, appeared left and right from fixation, followed by masks. The letter inside that shape matching the preceding cue had to be reported. In Experiment 1, either target, distractor, both, or no object matched the cue’s irrelevant color. Target-letter reports were most frequent in target-match trials and least frequent in distractor-match trials. Irrelevant cue and target color never matched in Experiment 2. Still, participants reported the distractor more often to the target’s disadvantage, when cue and distractor color matched. Thus, irrelevant features of a VWM template can influence covert attention in an involuntarily object-based manner when searching for trial-wise varying targets. Representations we are keeping briefly in memory can influence how we allocate our attention towards objects in the environment ,3,4,5,6.As theories and empirical evidence suggest that VWM maintains objects in the form of bound rather than segregated features ,26,27,28Gao and colleagues  delivereA problem with an active-passive WM status interpretation of the dual-task results is that it can never be predicted in advance, whether a VWM representation will be active or accessory during the interim search task or how many and which representations will be active  recruited at Bielefeld University, Germany, took part in the experiment after having provided written informed consent. Participants were naïve with respect to the study’s purpose, reported normal or corrected-to-normal visual acuity, and were compensated with 8€ per hour. The study was approved by the Committee for Ethics at the Department of Psychology (2015-024) at Bielefeld University in advance, and conducted in accordance with the Declaration of Helsinki.2, x = 0.3, y = 0.3). A black plus  with a size of 0.5 degrees of visual angle (dva) served as the central fixation marker. Real-world objects from the database of Konkle and colleagues ([http://cvcl.mit.edu/MM/objectCategories.html) were modified using MATLAB R2013b so that they extended 1.4 dva. in foveal vision (49 pixels) and filled with a single color  or red , and with one of 16 letters  superimposed on it in the background color  with a refresh rate of 100 Hz and a spatial resolution of 1024 × 768 pixels extending to 36 × 27 cm. Right gaze positions were recorded with an EyeLink 1000 desktop eye tracker  at 1000 Hz. A chin-and-forehead rest stabilized participants’ heads at a viewing distance of 71 cm. The experiment was programmed and run using SR Research’s Experiment Builder software on a Dell Precision T3600 with an NVIDIA GeForce GTX 970 graphics card. Luminance and color of all presented stimuli were measured at screen center in CIE Lxy coordinates with an X-Rite i1 Pro spectrophotometer. Stimuli were presented on a grey background  for a randomly chosen duration between 500 and 1000 ms (uniform distribution). In case of successful fixation, the letter display came on containing one colored object left and one colored object right from fixation at a distance of 4.3 dva for 170 ms duration. A letter was superimposed on each object in the background color . One objThe experiment consisted of four conditions . In the t-tests were used to compare the dependent variables in the experimental conditions to the no-match baseline in case of a significant ANOVA. We decided to test the three match conditions to the no-match condition because we think that the lack of any color repetition serves the best baseline performance, i.e., performance based on the shape signal alone without any preference for selecting either object based on color. A chance level of 0.05 was applied to all analyses.Data were analyzed and plotted using Excel 2010 (Microsoft) and SPSS Statistics 22 (IBM). The dependent variables were the percentages of reported targets and distractors. Kolmogorov-Smirnoff tests did not reveal any deviation from normal distribution. Repeated measures analyses of variance (ANOVA) were used to analyze whether the dependent variables differed significantly between the four conditions. Investigating target letter reports reveals whether target object processing is influenced by the different color-match conditions. Investigating distractor letter reports reveals whether target-distractor confusion is influenced by the different color-match conditions. If sphericity was violated, Greenhouse-Geisser epsilon is reported along with uncorrected degrees of freedom. Planned paired Raw data is provided in the F = 9.19, p < 0.01, pη2 = 0.38, ε = 0.50), as well as reported distractor letters  = 6.61, p < 0.01, pη2 = 0.31, ε = 0.54). Planned paired t-tests indicated that the effects were due to significantly more target letter reports (t(15) = 2.99, p < 0.01, zCohen’s d = 0.75) and less distractor letter reports (t(15) = 2.63, p < 0.05, zCohen’s d = 0.66) in case of the target color-match compared to the no-match baseline. Complementary, significantly less target letters (t(15) = 2.65, p < 0.05, zCohen’s d = 0.66) and more distractor letters (t(15) = 2.14, p < 0.05, zCohen’s d = 0.53) were reported in case of the distractor-match compared to the no-match baseline. Reports of the distractor letter (t(15) = 1.08, p = 0.30, zCohen’s d = 0.27) did not differ significantly between the both-match and the no-match condition, while target letter reports were marginally more frequent in the both-match than in the no-match condition (t(15) = 1.99, p = 0.06, zCohen’s d = 0.50). The mean values can be seen in Repeated measures ANOVAs indicated significant differences across conditions for the percent of reported target letters , green , red , or yellow . The procedure was exactly the same as in Experiment 1.A new sample of sixteen participants  recruited at Bielefeld University, Germany, took part in Experiment 2 after having provided written informed consent. Again, participants were naïve with respect to the study’s purpose, reported normal or corrected-to-normal visual acuity, and were compensated with 8€ per hour. The same materials were used as in Experiment 1, except that the vase and the pot could appear in one of four colors , namely t-tests compared the dependent variables between the distractor match and the no match condition. A chance level of 0.05 was applied.Experiment 2 consisted of two conditions . In the t-test revealed that participants reported significantly less targets (t(15) = 4.88, p < 0.01, zCohen’s d = 1.22) and more distractors (t(15) = 3.01, p < 0.05, zCohen’s d = 0.75) in case of the distractor color match compared to the no-match baseline. Descriptive values are provided in Raw data is provided in the In Experiment 2, participants reported more often the distractor instead of the target letter, when the distractor object matched the task-irrelevant color of the cue compared to the situation in which the cue’s color did not appear in the letter display. This was the case, although the target object never matched the cue’s color so that it is always dysfunctional to tune attention towards color-matching objects. The results of Experiment 2 thus indicate that the influence of the irrelevant feature of a trial-wise varying—and thus VWM-based—search template was not only object-based but also involuntary in nature that cannot be overcome by voluntary down-regulation.In the present study, we investigated whether a target object whose representation should be kept in VWM for the consecutive search task  will bias covert attention in an involuntarily object-based manner. In order to test this hypothesis, we investigated the influence of a task-irrelevant feature of a trial-wise varying search-target cue within a trailing partial report task that required to report a letter inside a target object in an unspeeded manner. The target letter in the target object was always accompanied by one distractor letter in a distractor object. Specifically, a colored shape cue (search target) indicated to participants on which of the following shapes a to-be-reported letter will appear. Although participants were told that the color was uninformative (Experiment 1) or even distractor-defining (Experiment 2), they could not prevent attending towards color-matching items in the search display so that they occasionally reported the letter on the cue-colored distractor to the disadvantage of the target letter. Thus, participants could neither ignore color nor use it as a template for rejection ,51. TogeThe results of the present study extend our previous finding that objects matching task-irrelevant features of a VWM object representation can capture the eyes—a proxy for covert attention —in a visThe results of the present study support the idea that VWM objects bias attention in an object-based manner. Results from dual-task studies investigating this question were inconclusive ,29. WhilAs stated above, the state of the VWM representation can often not be predicted in dual-task designs and is therefore inferred post-hoc from the results . By our single-task design , we ensuAlthough we think that an irrelevant feature of an active VWM object obligatorily elicits a bias signal, we also think that this does not guarantee a manifestation in observable behavior ,57. As iFinally, we want to address four open questions on the issue of involuntary top-down control by search irrelevant features of a trial-by-trial varying search/partial report target. Firstly, while several EEG and fMRI studies showed that the attentional capture is reflected in neuronal activity in case of behavioral effects ,62,63, n


In this paper, we demonstrate the laser-based gas sensing of methane near 3.3 µm inside hollow-core photonic crystal fibers. We exploit a novel anti-resonant Kagome-type hollow-core fiber with a large core diameter (more than 100 µm) which results in gas filling times of less than 10 s for 1.3-m-long fibers. Using a difference frequency generation source and chirped laser dispersion spectroscopy technique, methane sensing with sub-parts-per-million by volume detection limit is performed. The detection of ambient methane is also demonstrated. The presented results indicate the feasibility of using a hollow-core fiber for increasing the path-length and improving the sensitivity of the mid-infrared gas sensors. Infrared laser spectroscopy is a sensitive and selective technology with a number of practical applications. The simplest and frequently used solution for further improvement of the minimum detection limit (MDL) is to increase the sensing path-length using multi-pass cells. This approach typically leads to sensitivities at parts-per-million or parts-per-billion by volume levels  ,2,3,4,5.4) detection at 3.33 µm with sub-ppmv×m sensitivity is demonstrated. The obtained performance is almost an order of magnitude better (the improvement factor was approximately 8.22) comparing to the results obtained using the wavelength modulation spectroscopy technique [In this paper, we present high-resolution laser spectroscopy in the mid-infrared (mid-IR) region using Kagome-type negative curvature hollow-core PCF. This type of fiber has several advantages over PBG-PCFs. Its most important properties are reduced coupling between the cladding modes and the core modes and large core diameter. Using Kagome-type HC-PCF designed for mid-infrared wavelengths, an all-fiber difference frequency generation (DFG) source ,16 and cechnique , and it The experimental arrangement of the sensor setup is schematically shown in Coherent mid-infrared light was obtained through the difference frequency generation (DFG) process in a periodically poled lithium niobate (PPLN) crystal. Two distributed feedback (DFB) laser diodes with center wavelengths at 1063.5 nm and 1562 nm were used as seed sources for the DFG module. They were driven, and their temperatures were stabilized using commercially available controllers . Light from the seed sources was amplified up to 90 mW (at 1562 nm) and 80 mW (at 1063.5 nm) using standard erbium-doped and ytterbium-doped fiber amplifiers. Both wavelengths were combined using a fiber coupler. For optimum quasi-phase-matching, the DFG module was temperature-stabilized at 60 °C. With a highly efficient fiber-pigtailed DFG module, comprising a 40 mm-long PPLN waveguide crystal with a very high conversion efficiency (21%/W) [For filling the HCF with gas from a cylinder, an overpressure of ~850 Torr was used to ensure constant flow through the fiber. For filling with ambient air, a vacuum pump was used. The pressure inside the air-tight housing was reduced down to ~500 to ~600 Torr so that ambient air could be sucked through the front of the HCF. The flow rate was estimated to be between 100 and 320 µL/min. The beam exiting the fiber was focused onto a fast mercury-cadmium-telluride (MCT) detector .fm = 2.5 kHz was used, and variations of the frequency of the beat note were retrieved through the frequency demodulation at Ω and subsequent digital filtering of 2 × fm component [The detection technique implemented here was chirped laser dispersion spectroscopy, CLaDS. In contrast to typically used methods which target molecular absorption, CLaDS relies on measuring changes of the real part of the refractive index in the vicinity of absorption line ,18,21. Iomponent . The dem4 in nitrogen and the third measured with pure nitrogen. The wavelength modulation amplitude of ~5.8 GHz was determined experimentally to provide a maximal CLaDS signal amplitude (this was obtained through modulation of the laser current with a peak-to-peak amplitude of 15 mA). To enable recording the full CLaDS spectrum of the target transition, input light from the ~1 µm laser was additionally current-ramped at 25 mHz. A single data point was acquired within 100 ms. With an additional 60 ms needed for data processing, the resulting data acquisition rate was 6.25 Hz (250 points/spectrum). The spectra of methane were recorded for two different RF beat note powers at the input of the demodulator . This results in a small increase of noise but has no effect on the signal amplitude, which remained unchanged. There is also no baseline in the measured signals. −1. According to HITRAN, this line . This was confirmed with zero-gas analysis for which white noise-like performance is observed up to ~100 s . Allan deviation analysis indicates good stability of the setup. However, increasing the integration time above 200 s does not result in further improvement of the detection limit. This may be due to the presence of optical fringes in the system  or some thermal drifts in the setup.Thanks to the baseline-free nature of the CLaDS, continuous concentration monitoring could be performed without full spectral scanning. Instead, the frequency of the mid-infrared radiation could be adjusted to the peak of the CLaDS signal and only the signal amplitude could be measured . Figuree system . For sho2 measurement is (primarily) from ambient methane measured within the free-space section of the setup (approximately 60 cm). the standard deviation of the recorded data points is below 300 ppbv (for time series with 1 s averaging) which is consistent with detection limit estimated based on the Allan–Werle analysis. For system demonstration, HCF was alternately filled with nitrogen and laboratory air . The re4 but required a high power laser source to be coupled into the small diameter fiber. Here, the detection limit of 500 ppbv was obtained for 1 second averaging, using 1.3-m-long HCF. Ambient methane detection was also demonstrated (to our knowledge for the first time using hollow-core fiber as a gas cell). We have also shown that Kagome-type fiber  has sigIn this work, Chirped Laser Dispersion Spectroscopy was used for gas detection. This technique relies on the molecular dispersion measurement, not absorption-based sensing . The app